Following the discovery of Ras in 1981, a number of related small GTP-binding proteins (small GTPases) have been identified and their physiological functions have been extensively studied. These small GTPases (molecular mass 20-30 kDa) switch between the inactive GDP-bound state and the active GTP-bound state, a process that is highly regulated primarily by guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) (see, e.g., Hall, A., Science (1990) 249:635-640; Bourne, H. R. et al., Nature (1991) 349:117-127; each of which are herein incorporated by reference in their entireties).
To date, more than 50 different genes encoding small GTPases have been identified from yeast to mammals, forming the Ras superfamily. These small GTPases are largely divided into 5 families of Ras, Rho, Rab, Arf, and Ran, according to primary amino acid sequences and functional similarities. Of these, Rho, (Ras homologue) encodes a polypeptide having about 35% homology with Ras (see, e.g., Madaule, P., Cell (1985) 41:31-40; herein incorporated by reference in its entirety). The Rho family itself can be divided into 6 subfamilies based on primary amino acid sequence, structural motifs, and biological function, which includes the RhoA-related subfamily, Cdc42-related subfamily, Rac-related subfamily as well as the Rnd, RhoBTB and Miro subfamilies. Cellular activity of Rho has been studied by several methods including overexpression or microinjection of the active GTP-bound form of Rho to identify the phenotype of Rho activation. A second complimentary method has been to treat cells with botulinum C3 exoenzyme, which specifically ADP-ribosylates and inactivates endogenous Rho thereby identifying the phenotype of Rho inactivation (Narumiya, S. J Biochem (1996) 120:215-228). As such, Rho GTPases have been identified as key regulators of actin reorganization and have been implicated in the regulation of cell polarity, migration, cell shape, adhesion, contraction, as well as endo- and exocytosis (see e.g., Ridley, A. J., Trends Cell Biol (2001) 11:471-477).
Downstream targets of Rho GTPases that are involved in actin cytoskeletal reorganization include citron kinase, p140mDia, protein kinase N (PKN), p21-activated protein kinase (PAK), rhophillen, and rhotekin. The Rho-associated coiled-coil-forming protein kinases (ROCKs), first isolated by T. Ishizaki and coworkers in the mid-1990s, were the first and best characterized effectors of RhoA and were initially characterized for their roles in mediating the formation of RhoA-induced stress fibers and focal adhesion through their effects on the phosphorylation of myosin light chain (Matsui, T., et. al., EMBO J. (1996) 15:2208-2216; Leung, T., et. al., Mol. Cell. Biol. (1996) 16:5315-5327). Subsequently, ROCKs have been shown to play a role in many key cellular functions such as cell motility, invasion, contraction, differentiation, migration, and survival (Riento, K., Ridley, A., Nature Rev. Mol. Cell. Biol. (2003) 4:446-456).
ROCKs are serine/threonine protein kinases with a molecular mass of approximately 160 kDa. Two isoforms encoded by two different genes have been identified: ROCKI (also known as ROKβ or p160ROCK) and ROCKII (or ROKα). The isoforms share an overall amino acid sequence identity of 65% and 92% sequence identity in their kinase domains. ROCKs are most homologous to members of the AGC kinases such as myotonic dystrophy kinase (DMPK), DMPK-related cell division control protein 42 (Cdc42)-binding kinase (MRCK), and citron kinase (CK). In general, this family of kinases consists of an amino-terminal kinase domain followed by a coiled-coil-forming region and then a pleckstrin-homology (PH) domain with an internal cysteine-rich repeat at the carboxy-terminal. In addition, ROCKs also contain a Rho-binding domain (RBD) within their coiled-coil domain. In the inactive state, the carboxy-terminal domains bind to the amino-terminal region, which forms an autoinhibitory loop. Activated, GTP-bound Rho binds to the RBD of ROCK, which results in an open conformation of the kinase thereby freeing the catalytic activity. ROCKs can also be activated by lipid binding (e.g., arachidonic acid and sphingosylphosphorylcholine) to the PH domain. ROCK activity can also be induced during apoptosis as caspase 3 can cleave the auto-inhibitory loop of ROCKI while granzyme B and caspase 2 cleave ROCKII in a similar fashion, both of which result in constitutively active ROCK.
In response to activators of Rho, such as lysophosphatidic acid (LPA) or sphingisone-1 phosphate (S1P), which stimulate Rho GEFs and lead to the formation of active GTP-bound Rho, ROCKs mediate a broad range of cellular responses involving the actin cytoskeleton through phosphorylation of a variety of cellular targets. For example, phosphorylation of the motor protein myosin II has an important role in regulating actomyosin contractility. ROCK can directly phosphorylate myosin light chain (MLC), which results in subsequent myosin-actin interactions and enhanced cell contractility. ROCK can also indirectly regulate MLC phosphorylation levels through phosphorylation (and inactivation) of myosin light chain phosphatase (MLCP). Another downstream target of ROCK are LIM kinases 1 and 2, whose phosphorylation leads to inhibition of cofilin-mediated actin-filament disassembly and therefore an increase in the number of actin filaments. Other cellular targets of ROCK include the ezrin/radixin/moesin (ERM) protein complex, intermediate filament proteins such as vimentin, and the filamentous (F)-actin-binding protein adducin (Riento, K., Ridley, A., Nature Rev. Mol. Cell. Biol. (2003) 4:446-456).
Despite having similar kinase domains, ROCK1 and ROCK2 may have different cellular functions and have different downstream targets. For example, in vitro ROCK1 has been shown to phosphorylate LIM kinase 1 and 2, while ROCK 2 phosphorylates MLC, adducin, smooth muscle-specific basic calponin, and collapsing response mediator protein-2 (CRMP2), a neuronal protein that is involved in LPA-induced collapse of growth cones (Riento, K., Ridley, A., Nature Rev. Mol. Cell. Biol. (2003) 4:446-456). Furthermore, siRNA experiments have demonstrated distinct roles for ROCK1 and ROCK2 in rat embryonic fibroblast cells where ROCK1 was important for stress fiber formation and stabilization of focal adhesion sites, while ROCK2 activity was involved in phagocytosis of matrix-coated beads (Yoneda, A., et. al., J. Cell Biol. (2005) 170:443-453). Differential expression and regulation in various cell types has also been observed. For example, only ROCK1 is cleaved by caspase 3 during apoptosis while ROCK2 is cleaved by granzyme B and caspase 2. In addition, ROCK1 expression tends to be more ubiquitous, while ROCK2 is most highly expressed in muscle and brain tissues indicating that the protein may have a specialized role in these cell types (Nakagawa, O., et. al., FEBS Lett. (1996) 392:189-193). However, in vivo data relating ROCK1 and ROCK2 isoforms to differential functions is still lacking.
Abnormal activation of the Rho/ROCK pathway has been shown to play a role in a wide range of diseases, both in those involving abnormal smooth muscle tone or smooth muscle hyperreactivity as well as in pathological processes involving non-smooth muscle cells. For example, Rho/ROCK mediated-signaling has been shown to be involved in the pathogenesis of hypertension, vasospasms leading to vasoconstriction and ischemia (both cerebral and coronary), bronchial asthma, preterm labor, erectile dysfunction, and glaucoma (Werrschureck, N., Offermanns, S., J Mol. Med. (2002) 80:629-638 and references therein). Vascular diseases such as hypertension, atherosclerosis, postangioplasty restenosis, and transplant arteriosclerosis, which are characterized by abnormal vascular smooth muscle cell (VSMC) proliferation and migration have also been shown to be associated with increased Rho/ROCK signaling. Rho/ROCK mediated signaling is also associated with disease in non-smooth cells such as myocardial hypertrophy. Abnormal activation of the Rho/ROCK pathway has been observed in various disorders of the central nervous system (CNS; Mueller, B. K. et al., Nature Rev. Drug Discovery (2005) 4:387-398 and references therein). Injury to the adult vertebrate brain and spinal cord activates ROCKs, thereby inhibiting neurite growth and sprouting. As such, there is significant potential therapeutic use of ROCK inhibitors for the treatment of various neurological disorders, including spinal-cord injury, Alzheimer's disease, stroke, multiple sclerosis, and neuropathic pain. Furthermore, tumor cell migration and invasion involves Rho-mediated processes and activation of RhoA or of ROCK has been shown to increase the invasiveness of cultured rat hepatoma cells (Itoh, K., et al., Nat. Med. (1999) 5:221-225). In addition, a number of oncogenes encode exchange factors for Rho suggesting that the Rho/ROCK pathway is an attractive candidate for new anticancer strategies.
Given the extensive involvement of the Rho/ROCK pathway in many disease states, there has been considerable interest in the development of ROCK inhibitors in the last 20+ years. Fasudil
and Y-27632
were the first small-molecule ROCK inhibitors discovered (Uehata, M. et al. Nature (1997) 389:990-994). Subsequently, many more inhibitors have been developed and can be generally grouped into four classes according to their hinge-binding scaffold: isoquinolines (e.g, fasudil), 4-aminopyridines (e.g., Y-27632), indazoles, and amide and urea derivatives. ROCK inhibitors reported to date act by competitive interaction at the ATP binding site. However, due to the high sequence homology between ATP-binding sites, the development of inhibitors specific for ROCK has been challenging. Although few results have been reported for ROCK inhibitors in general, data reported for Y-27632 and fasudil demonstrate some cross-reactivity of these inhibitors against other kinases. For example, Y-27632 showed selectivity against 21 of 25 kinases tested but inhibited protein kinase N (PKN or PRK2) with equal potency and was only 10-50-fold selective over mitogen- and stress-induced kinase 1 (MSK1), mitogen-activated protein kinase-activated protein kinase 1b (MAPKAPK1b), citron kinase, and phosphorylase kinase (PHK) (Davies, S. P., et al. Biochem J (2000) 351:95-105). In the same study, fasudil was shown to be less selective that Y-27632 showing selectivity against only 19 of the 27 kinases tested. Furthermore, Y-27632 and fasudil (similar to other reported ROCK inhibitors) do not demonstrate any ROCK isoform selectivity with almost identical inhibition of ROCK1 and ROCK2. Although animal studies involving ROCK1 and ROCK2 knock-out mice suggest distinct physiological roles for the two ROCK isoforms, data is still lacking. However, currently available ROCK inhibitors cannot be used to differentiate the role of ROCK1 versus ROCK2 either in cellular signaling or substrate recognition, or more importantly, in the specific role of each isoform in disease.
Fasudil has been marketed in Japan since 1995 for the treatment of vasospasm after subarachnoid hemorrage and safety profile data indicate that it is well tolerated in humans. It has been shown to have beneficial effects in a number of cardiovascular diseases including angina pectoris, hypertension, coronary vasospasm, restenosis after percuteneous coronary intervention, and arteriosclerosis (Hirooka, Y., Shimokawa, H., Am. J. Cardiovasc. Drugs (2005) 5:31-39 and references therein). Y-27632 has been much less investigated in vivo but limited studies have demonstrated that (similar to fasudil) it is rapidly metabolized and brain penetration may be too low to achieve therapeutic levels for CNS diseases. In addition, both inhibitors, like other ATP-competitive inhibitors, demonstrate a 100-1.000-fold decrease in activity in cellular assays, as compared to in vitro activities due to competition with intracellular micromolar ATP concentrations. At such a high cellular concentration, their low-to-moderate kinase selectivity for PKN, citron kinase, MSK1, and MAPKAPK1b can lead to additional off-target effects. As such, the development of a new structural class of ROCK inhibitors may provide more selective ROCK inhibitors against other kinases as well as the development of ROCK isoform-specific inhibitors. Such inhibitors have the potential to be used therapeutically in both cancer and heart disease given the evidence from animal studies of the involvement of ROCK in invasion, metastasis, neuroregeneration, and smooth muscle-cell contraction.
What are needed are improved compositions and methods for inhibiting Rho kinase activity in subjects afflicted with diseases and conditions associated with aberrant Rho kinase activity.